This project will investigate the cell and molecular biology of sialyltransferases. In the past project period a cDNA containing the full length coding sequence of a Beta-galactoside alpha 2,6 sialyltransferase was obtained. Two other sialyltransferase cDNAs will be cloned, a beta-galactoside alpha 2,3 sialyltransferase, using degenerate oligonucleotide probes derived from peptide sequence, and an N-acetylgalactosaminyl alpha 2,6 sialyltransferase, using antibody to screen and expression library. It is anticipated that the comparison of the sequences of the three sialyltransferases will reveal homologies that define a sialyltransferase gene family. Constant domain oligonucleotide probes will be used to screen lambda gt11 libraries at low homology for other sialyltransferase cDNAs. Sialyltransferase cDNAs containing the coding sequence of the enzymes will be inserted into the pMSG and pECE vectors and expressed in CHO cells under the early SV40 promoter. CHO clones expressing high levels of sialyltransferase will be isolated for the purpose of producing large amounts of sialyltransferase. This will solve a current shortage of these enzymes for use as enzymatic reagents in the analysis of the biological roles of sialic acids. A long term goal is to develop general methodologies for the cloning and expression of glycosyltransferases, making them available as reagents for glycoconjugate research. The cell biology and regulation of expression of sialyltransferases will also be examined, beginning with the beta-galactoside alpha 2,6 sialyltransferase for which a cDNA is already available. The basis for localization of the enzyme in the trans region of the Golgi apparatus of cells will be examined by site directed mutagenesis of the cytoplasmic, transmembrane, and lumenal stem domains. Results of the mutagenesis will be evaluated by expression in COS cells using the pSVL vector. Sialyltransferase will be localize by immunofluorescence and immunoelectron microscopy, as well as by biochemical correlated. Preliminary studies using Northern blots show that the sialyltransferase exhibits dramatic (50 fold) differences in mRNA levels, suggesting tissue specific transcriptional control. In addition, the 5' untranslated leader of the sialyltransferase cDNA exhibits several ATGs which are in excellent context for translation initiation start sites, and are likely involved in translational regulation. Due to these observation, investigations into the transcriptional and translational control of sialyltransferase gene expression will be initiated. These combined studies are expected to yeild important new information on regulation and organization of the cellular glycoyslation machinery.